Use of non-toxic crosslinking reagents to improve fatigue resistance and reduce mechanical degradation of intervertebral disc and other collagenous tissues

ABSTRACT

A method of improving the resistance of collagenous tissue to mechanical degradation in accordance with the present invention comprises the step of contacting at least a portion of a collagenous tissue with an effective amount of a crosslinking reagent. The crosslinking reagent includes a crosslinking agent such as genipin and/or proanthrocyanidin. Further, the crosslinking reagent may include a crosslinking agent in a carrier medium. The collagenous tissue to be contacted with the crosslinking reagent is preferably a portion of an intervertebral disc or articular cartilage. The contact between the tissue and the crosslinking reagent is effected by injections directly into the select tissue using a needle. Alternatively, contact between the tissue and the crosslinking reagent is effected by placement of a time-release delivery system such as a gel or ointment, or a treated membrane or patch directly into or onto the target tissue. Contact may also be effected by, for instance, soaking.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/316,287, filed Aug. 31, 2001.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method for treatment oftissue, for example, collagenous tissue, where a deleterious mechanicalloading environment contributes to the degradation of the tissue. Morespecifically, the present invention relates to a method for treatment ofdegenerated intervertebral discs to improve fatigue resistance, and tonon-toxic crosslinking reagents that are effective fatigue inhibitors.

[0004] 2. Description of the Related Art

[0005] Back pain and disability associated with spinal degeneration andinstability continue to be one of the costliest and most prevalenthealth problems in western civilization. Current treatments for spinalinstability and low-back pain, including spinal fusion, are generallyineffective in slowing the progression of degeneration. Epidemiologicaland morphological studies have shown that the capacity of spinal tissueto withstand repetitive loading is one critically important factor inthe progression of spinal osteoarthritis (Magora 1972, Kelsey 1975,Frymoyer 1983, Videman 1990).

[0006] The organization of collagen and proteoglycans within theintervertebral disc plays an important role in determining thebiomechanical properties of the disc. Biochemical alterations in thestructure of the annular matrix affect the disc's durability, that is,its ability to withstand repetitive mechanical loading. Previous studieshave shown that nonreducible pyridinoline cross links are predominant inadult cartilage, bone, and intervertebral discs and these collagencrosslinks are thought to be critical for the structural integrity(enzymatic and mechanical) of adult connective tissue (Burgeson andNimni, 1992, Eyre, 1988). Pentosidine crosslinking has been shown toincrease with age in articular cartilage and intervertebral discs (Bank1998, Pokharna 1998).

[0007] A role for naturally occurring crosslinks in stabilizingdegenerating discs has been suggested. Duance (1998) noted that whilethe nonenzymic derived crosslink pentosidine showed an expected agerelated increase, its level was lower in the more severely degeneratedsamples. It may be that age related tissue changes—i.e. micro-damageaccumulation—combined with inadequate levels of crosslinks made thesediscs more vulnerable to mechanical degradation. Age related crosslinks(pentosidine) have been shown to increase the strength and stiffness ofarticular cartilage (Chen 2001) while age related microdamageaccumulation would act to decrease strength and stiffness. With regardto viscoelastic properties, Lee (1989) found that aldehyde fixation(crosslinking) reduced stress-relaxation and creep in bovinepericardium, while fatigue loading produced an increase instress-relaxation and creep in our preliminary testing of intervertebraldiscs.

[0008] Crosslinking reagents are capable of improving the tensileproperties of collagen-based biomaterials. Osborne et al (1998) foundmechanical strength of acellular collagen gels was most improved using acombination of crosslinking agents. Other researchers have also foundthat crosslinking treatments can increase the strength of collagenoustissues (Wang 1994, Chachra 1996, Sung 1999, Zeeman 1999). Sung (1999)found that a naturally occurring cross linking agent, genipin, providedgreater ultimate tensile strength and toughness when compared with othercrosslinking reagents. Genipin also demonstrated significantly lesscytotoxicity compared to other more commonly used crosslinking agents.However it also stood out in a negative sense with regard to eliminatingtissue anisotropy in bovine pericardium. Several researchers have statedtheir expectation that crosslinking of collagenous tissue may make thetissue more prone to fatigue failure (Bank 1998, Chen 2001, Kerin 2001).However, it is believed that the opposing view—that crosslinkingcollagenous tissue may actually benefit fatigue resistance—has not beenrecorded in the medical literature. It is believed that collagencrosslinks may act as sacrificial bonds to protect collagenous tissuesby dissipating energy and improving fatigue resistance.

[0009] Fatigue is a weakening of a material due to repetitive appliedstress. Fatigue failure is simply a failure where repetitive stresseshave weakened a material such that it fails below the original ultimatestress level. In bone, two processes—biological repair and fatigue—arein opposition, and repair generally dominates. In the intervertebraldisc, the prevalence of mechanical degradation of the posterior annulus(Osti 1992) suggests that fatigue is the dominant process. Active tissueresponse (adaptation, repair) does not play a strong role in the case ofmature intervertebral disc annular material. As a principally avascularstructure, the disc relies on diffusion for nutrition of its limitednumber of viable cells. Age related changes interfere with diffusionpresumably contributing to declining cell viability and biosyntheticfunction (Buckwalter et al. 1993, Buckwalter 1995). Age related declinein numbers of cells and cell functionality compromises the ability ofthe cells to repair mechanical damage to the matrix. While regenerationof the matrix in the nucleus following enzymatic degradation has beenaccomplished, albeit inconsistently (Deutman 1992), regeneration offunctional annular material has not yet been realized.

[0010] Combined with this limited potential for repair or regeneration,studies have shown that posterior intervertebral disc tissue isvulnerable to degradation and fatigue failure when subjected tonon-traumatic, physiologic cyclic loads. Prior work has showndeterioration in elastic-plastic (Hedman 99) and viscoelastic (Hedman00) material properties in posterior intervertebral disc tissuesubjected to moderate physiological cyclic loading. Cyclic loadmagnitudes of 30% of ultimate tensile strength produced significantdeterioration of material properties with as little as 2000 cycles.Green (1993) investigated the ultimate tensile strength and fatigue lifeof matched pairs of outer annulus specimens. They found that fatiguefailure could occur in less than 10,000 cycles when the vertical tensilecyclic peak exceeded 45% of the ultimate tensile stress of the matchedpair control. In addition, Panjabi et al (1996) found that single cyclesub-failure strains to anterior cruciate ligaments of the knee alter theelastic characteristics (load-deformation) of the ligament. Osti (1992)found that annular tears and fissures were predominantly found in theposterolateral regions of the discs. Adams (1982) demonstrated thepropensity of slightly degenerated discs to prolapse posteriorly whenhyperflexed and showed that fatigue failure might occur in lumbar discsas the outer posterior annulus is overstretched in the verticaldirection while severely loaded in flexion. In an analytical study,interlaminar shear stresses, which can produce delaminations, have beenfound to be highest in the posterolateral regions of the disc (Goel1995). These prior data indicate: 1) the posterior disc and posteriorlongitudinal ligament are at risk of degenerative changes, and that 2)the mechanism of degeneration can involve flexion fatigue.

[0011] To date, however, no treatments capable of reducing mechanicaldegradation to collagenous tissues currently exist. In fact, no othercollagenous tissue fatigue inhibitors have been proposed. A needtherefore exists for a method for improving the resistance ofcollagenous tissues in the human body to fatigue and for reducing themechanical degradation of human collagenous tissues, in particular, theposterior annulus region of the intervertebral disc.

SUMMARY OF THE INVENTION

[0012] It is one object of the present invention to provide a method ofimproving the resistance of collagenous tissues in the human body tomechanical degradation by contacting the tissue with crosslinkingreagents.

[0013] It is another object of the present invention to provide a methodof curtailing the progressive mechanical degradation of intervertebraldisc tissue by enhancing the body's own efforts to stabilize aging discsby increasing collagen crosslinks.

[0014] It is another object of the present invention to provide a methodthat uses crosslinking reagents with substantially less cytotoxicitycompared to common aldehyde fixation agents in order to facilitatedirect contact of these reagents to tissues in the living human body.

[0015] It is another object of the present invention to increase thecrosslinking of disc annular tissue by directly contacting living humandisc tissue with appropriate concentrations of a non-toxic crosslinkingreagent (or a mixture of crosslinking reagents) such as genipin (ageniposide) or proanthrocyanidin (a bioflavonoid).

[0016] It is another object of the present invention to provide atreatment method for minimally invasive delivery of the non-cytotoxiccrosslinking reagent such as injections directly into the select tissueusing a needle or placement of a time-release delivery system such as acarrier gel or ointment, or a treated membrane or patch directly into oronto the target tissue.

[0017] It is another object of the present invention to a compositioncomposed of non-toxic crosslinking reagents that can be used aseffective fatigue inhibitors.

[0018] In accordance with the present invention, there is provided amethod for treatment of tissues where a deleterious mechanical loadingenvironment contributes to the degradation of the tissue. Thedeleterious mechanical loading environment may consist of normalphysiological repetitive loading, otherwise known as fatigue. Thepresent invention provides a method for treatment of degeneratedintervertebral discs to improve fatigue resistance. The presentinvention also provides non-toxic crosslinking compositions that areeffective fatigue inhibitors.

[0019] A method of improving the resistance of collagenous tissue tomechanical degradation in accordance with the present inventioncomprises the step of contacting at least a portion of a collagenoustissue with an effective amount of a crosslinking reagent. Thecrosslinking reagent includes a crosslinking agent such as genipinand/or proanthrocyanidin. Further, the crosslinking reagent may includea crosslinking agent in a carrier medium. The collagenous tissue to becontacted with the crosslinking reagent is preferably a portion of anintervertebral disc or articular cartilage. The contact between thetissue and the crosslinking reagent is effected by injections directlyinto the select tissue using a needle. Alternatively, contact betweenthe tissue and the crosslinking reagent is effected by placement of atime-release delivery system such as a gel or ointment, or a treatedmembrane or patch directly into or onto the target tissue. Contact mayalso be effected by, for instance, soaking or spraying.

DESCRIPTION OF THE FIGURES

[0020]FIG. 1 is a graph of relaxation (N) v. numbers of cycles showingthe effect of genipin crosslinking treatments (G1=0.033 g/mol, G2=0.33g/mol) on posterior intervertebral disc stress relaxation.

[0021]FIG. 2 is a graph of Brinnell's hardness index v. numbers ofcycles showing the effect of genipin crosslinking treatments (G1=0.033g/mol, G2=0.33 g/mol) on posterior intervertebral disc hardness orresistance to penetration.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides a method of improving theresistance of collagenous tissues in the human body to mechanicaldegradation comprising the step of contacting at least a portion of acollagenous tissue with an effective amount of a crosslinking reagent.The method of the present invention also provides a method of curtailingthe progressive mechanical degradation of intervertebral disc tissue byenhancing the body's own efforts to stabilize aging discs by increasingcollagen crosslinks. This mechanical degradation may be in response tophysiologic levels of repetitive loading.

[0023] The crosslinking reagent of the present invention is notparticularly limited. Any crosslinking reagent known to be substantiallynon-cytotoxic and to be an effective cross-linker of collagenousmaterial may be used. The crosslinking reagent is required to besubstantially non-cytotoxic in order to facilitate direct contact of thecrosslinking agent to tissues in the living human body. Preferably, thecrosslinking reagent exhibits substantially less cytotoxicity comparedto common aldehyde fixation agents. More preferably, a non-cytotoxiccrosslinking reagent is used.

[0024] The crosslinking reagent includes at least one crosslinkingagent. The crosslinking agent chosen in accordance with the presentinvention is an effective cross-linker of collagenous material. Whenused in a cross-linking reagent, an effective crosslinker is one thatincreases the number of crosslinks in the collagenous tissue when thecrosslinker is brought into contact with a portion of the collagenoustissue. An effective crosslinker-improves the fatigue resistance of thetreated tissue, reduces material property degradation resulting fromrepetitive physiologic loading, or reduces the increase of viscoelasticproperties of the treated tissue due to fatigue loading. Likewise, aneffective crosslinker may reduce the decrease in elastic-plasticproperties due to fatigue loading of the treated tissue. In oneembodiment of the present invention, the crosslinking agent is Genipin,a non-toxic, naturally occurring crosslinking agent. Genipin is obtainedfrom its parent compound, geniposide, which may be isolated from thefruits of Gardenia jasminoides. Genipin may be obtained commerciallyfrom Challenge Bioproducts Co., Ltd., 7 Alley 25, Lane 63, TzuChiang St.404 Taichung Taiwan R.O.C., Tel 886-4-3600852. In another embodiment ofthe present invention, the crosslinking agent is a bioflavonoid, andmore specifically, the bioflavonoid is proanthrocyanidin. A mixturecontaining proanthrocyanidin can be obtained as MegaNatural™ Gold fromPolyphenolics, Inc, 22004 Rd. 24, Medera, Calif. 93638, Tel559-637-5961. More than one crosslinking agent may be used.

[0025] The crosslinking reagent may include a carrier medium in additionto the crosslinking agent. The crosslinking agent may be dissolved orsuspended in the carrier medium to form the crosslinking reagent. In oneembodiment, a crosslinking agent is dissolved in a non-cytotoxic andbiocompatible carrier medium. The carrier medium is required to besubstantially non-cytotoxic in order to mediate the contact of thecrosslinking agent to tissues in the living human body withoutsubstantial damage to the tissue or surrounding tissue. Preferably, thecarrier medium chosen is water, and more preferably, a saline solution.Preferably, the pH of the carrier medium is adjusted to be the same orsimilar to the tissue environment. Even more preferably, the carriermedium is buffered. In one embodiment of the present invention, thecarrier medium is a phosphate buffered saline (PBS).

[0026] When the crosslinking agent is dissolved in a carrier medium, theconcentration of the crosslinking agent in the carrier medium is notparticularly limited. The concentration may be in any amount effectiveto increase the crosslinking of the tissue while at the same timeremaining substantially noncytotoxic. When the crosslinking agent isgenipin, the concentration of the crosslinking agent is preferablygreater than 0.033% in PBS (wt %), and more preferably, about 0.33% inPBS (wt %).

[0027] In accordance with the present invention, the crosslinkingreagent is brought into contact with a portion of a collagenous tissue.As used herein, collagenous tissue is defined to be a structural or loadsupporting tissue in the body comprised of a substantial amount ofcollagen. Examples would include intervertebral disc, articularcartilage, ligament, tendon, bone, and skin. In general, the portion ofthe collagenous tissue to be brought into contact with the crosslinkingreagent is the portion of the tissue that is subject to loading.Further, where at least some degradation of the collagenous tissue hasoccurred, the portion of the tissue to be contacted with thecrosslinking reagent is at least the portion of the tissue that has beendegraded. Preferably, the entire portion that is subject to loading orthe entire portion that is degraded is contacted with the crosslinkingreagent. Further, the tissue adjacent the portion of collagenous tissuesubject to the loading may also be contacted with the crosslinkingreagent.

[0028] The collagenous tissues that are particularly susceptible for usein accordance with the present invention include intervertebral discsand articular cartilage. Where the collagenous tissue is anintevertebral disc, the portion of the intervertebral disc that ispreferably contacted by the crosslinking reagent is the posterior andposterolateral annulus fibrosis.

[0029] The selected portion of the collagenous tissue must be contactedwith an effective amount of the non-toxic crosslinking reagent. An“effective amount” is an amount of crosslinking reagent sufficient tohave a mechanical effect on the portion of the tissue treated.Specifically, an “effective amount” of the crosslinking reagent is anamount sufficient to improve the fatigue resistance of the treatedtissue, reduce material property degradation resulting from repetitivephysiologic loading, or reduce the increase of viscoelastic propertiesof the treated tissue due to fatigue loading, or reduce the decrease ofelastic-plastic properties of the treated,tissue due to fatigue loading.An effective amount may be determined in accordance with theviscoelastic testing and/or the elastic-plastic testing described hereinwith respect to Examples 1 and 2.

[0030] The method of the present invention includes contacting at leasta portion of the collagenous tissue with an effective amount of thecrosslinking reagent. The contact may be effected in a number of ways.Preferably, the contacting of collagenous tissue is effected by a meansfor minimally invasive delivery of the non-cytotoxic crosslinkingreagent. Preferably, the contact between the tissue and the crosslinkingreagent is effected by injections directly into the select tissue usinga needle. Preferably, the contact between the tissue and thecrosslinking reagent is effected by injections from a single or minimumnumber of injection locations. Preferably, an amount of crosslinkingsolution is injected directly into the targeted tissue using a needleand a syringe. Preferably, a sufficient number of injections are madealong the portion of the tissue to be treated so that complete coverageof the portion of the collagenous tissue to be treated is achieved.

[0031] Alternatively, contact between the tissue and the crosslinkingreagent is effected by placement of a time-release delivery systemdirectly into or onto the target tissue. One time-released deliverysystem that may be used is a treated membrane or patch. Areagent-containing patch may be rolled into a cylinder and insertedpercutaneously through a cannula to the tissue sight, unrolled and usinga biological adhesive or resorbable fixation device (sutures or tacks)be attached to the periphery of the targeted tissue.

[0032] Another time-released delivery system that may be used is a gelor ointment. A gel or ointment is a degradable, viscous carrier that maybe applied to the exterior of the targeted tissue.

[0033] Contact also may be effected by soaking or spraying, such asintra-capsular soaking or spraying, in which an amount of crosslinkingsolutions could be injected into a capsular or synovial pouch.

[0034] It should be noted that the methods and compositions treatedherein are not required to permanently improve the resistance ofcollagenous tissues in the human body to mechanical degradation.Assuming that a person experiences 2 to 20 upright, forward flexionbends per day, the increased resistance to fatigue associated withcontact of the collagenous tissue with the crosslinking reagent, may,over the course of time, decrease. Preferably, however, the increasedresistance to fatigue lasts for a period of several months to severalyears without physiologic mechanical degradation. Under suchcircumstance, the described treatment can be repeated at the timeperiods sufficient to maintain an increased resistance to fatigueresistance. Using the assumption identified above, the contacting may berepeated periodically to maintain the increased resistance to fatigue.For some treatment, the time between contacting is estimated tocorrespond to approximately 1 year for some individuals. Therefore, witheither a single treatment or with repeated injections/treatments, themethod of the present invention minimizes mechanical degradation of thecollagenous tissue over an extended period of time.

EXAMPLES 1 and 2

[0035] Thirty-three lumbar intervertebral joints were obtained from tenfour-month-old calf spines. The intervertebral joints were arbitrarilydivided into 3 groups: untreated controls-12 specimens, Genipintreatment 1 (G1)-6 specimens, and Genipin treatment 2 (G2)-13 specimens.The G1 treatment involved 72 hours of soaking the whole specimen in PBSwith a 0.033% concentration of Genipin. Similarly the G2 treatmentinvolved 72 hours of soaking whole specimens in PBS with 0.33%concentration of Genipin. 0.33% Genipin in PBS is produced by dilutionof 50 ml of 10×PBS (Phosphate Buffered Saline) with distilled water by afactor of 10 to give 500 ml (500 gm) of PBS and mixing in 1.65 grams ofgenipin to produce the 0.33% (wt %, gm/gm) solution. Previous testingwith pericardium and tendon tissue samples demonstrated the reduction oftissue swelling (osmotic influx of water into the tissue) resulting fromcrosslinking the tissue. Some controls were not subjected to soakingprior to fatigue testing. Others were soaked in a saline solution for 72hours. Water mass loss experiments were conducted to establish theequivalency of outer annulus hydration between the genipin soaked and0.9% saline soaked controls. The selection of treatments was randomizedby spine and level. The vertebral ends of the specimens were then pottedin polyurethane to facilitate mechanical testing.

[0036] Indentation testing and compression/flexion fatigue cycling werecarried out in the sequence presented in Table 1. TABLE 1 Experimentalprotocol Measurement Sequence Measurement Location 1 Stress Center ofthe Posterior Annulus Relaxation 2 Hardness Center of the PosteriorAnnulus 3000 Compression/Flexion Fatigue Cycles 3 Stress 4 mm Lateral toCenter Relaxation 4 Hardness Center of the Posterior Annulus Additional3000 Compression/Flexion Fatigue Cycles 5 Stress 4 mm Lateral to Center(Opposite Side) Relaxation 6 Hardness Center of the Posterior Annulus

[0037] At the prescribed points in the loading regimen, indentationtesting was used to find viscoelastic properties as follows. Stressrelaxation data was gathered by ramp loading the 3 mm diameterhemispherical indenter to 10 N and subsequently holding thatdisplacement for 60 s, while recording the resulting decrease in stress,referred to as the stress relaxation. Indentation testing was alsoutilized to determine elastic-plastic properties by calculating ahardness index (resistance to indentation) from ramp loading data. Priorto recording hardness measurements, the tissue is repeatedly indented 10times (60 s/cycle, to the displacement at an initial 10 N load).

[0038] This test protocol is based on two principles. First,viscoelastic effects asymptotically decrease with repeated loading.Secondly, hardness measurements are sensitive to the loading history ofthe tissue. However this effect becomes negligible following 10 loadingcycles. In order to minimize these effects, viscoelastic data (stressrelaxation) was collected from tissue that had not previously beenindented. Alternately, elastic-plastic data (hardness) was collectedfrom tissue that had been repeatedly loaded (preconditioned). In thiscase, repetitive indentation was intended to reduce the undesiredeffects of the changing viscoelastic properties, namely lack ofrepeatability, on hardness measurements. These testing procedures werederived from several preliminary experiments on the repeatability of themeasurements with variations of loading history and location.

[0039] Following initial indentation testing, the specimen was loadedrepetitively in flexion-compression at 200 N for 3000 cycles at a rateof 0.25 Hz. The load was applied perpendicularly to the transverseplane, 40 mm anterior to the mid-point of the specimen in the transverseplane. A second set of indentation testing data is then collectedfollowing fatigue cycling. This procedure was followed for two fatigueloading cycles. During all testing, the specimens were wrapped in salinewetted gauze to maintain their moisture content. Fatigue cycling andnon-destructive indentation testing were carried out on an MTS 858.02biaxial, table-top, 10 kN capacity servo-hydraulic materials teststation (MTS, Eden Prairie, Minn.), with the MTS Test Star dataacquisition system. Several statistical measures were calculated toevaluate the significance of the results. A nested two-way analysis ofvariance (ANOVA) was utilized to confirm effects due to treatment andnumber of fatigue cycles. Due to the non-parametric nature of the data,the Mann-Whitney non-parametric rank-sum test was used to assess thenull hypotheses that the treatment did not affect: 1) the pre-cyclingmechanical parameters of the tissue, or 2) the amount of change(degradation) in elastic-plastic and viscoelastic mechanical parametersdue to fatigue loading. The confidence level for statisticalsignificance was set at p<0.05.

[0040] Nested two-way ANOVA analysis determined that both viscoelastic(relaxation) and elastic-plastic (hardness) mechanical parameters wereindependently affected by fatigue cycling and by treatment type. Thesestatistical results are presented in Table 2.

[0041] The relaxation test results are presented graphically in FIG. 1.There was an initial shift downward of the relaxation curve caused bythe crosslinking treatment. This would represent a beneficial effect ashigher stress relaxation would be associated with more severely degradedtissue (Lee 1989). The initial pre-fatigue relaxation of the G1 and G2treatment groups were 26% and 19% less than (p=0.009 and p=0.026) thepre-fatigue relaxation of the controls respectively. There was alsodramatic improvement in fatigue resistance as demonstrated by the changein relaxation after 6000 non-traumatic loading cycles. The change inrelaxation due to 6000 fatigue cycles for the G2 treated discs was lessthan a third of the change in the controls (p=0.044). However, thelesser concentration of Genepin did not bring about the same improvementin fatigue resistance.

[0042] The hardness test results are presented graphically in FIG. 2.There is an initial shift upward of the hardness data caused by the G2crosslinking treatment. This would represent a beneficial effect as lossof hardness would signal a loss of structural integrity in the tissue.The initial pre-fatigue hardness of the G2 treatment group was 17%greater than that of the control group (p=0.026). However thisbeneficial effect appears to have eroded prior to 3000 fatigue cyclesand the change in hardness between 3000 and 6000 cycles is essentiallythe same for the two groups (G2=−0.94, Control=−1.01). TABLE 2 Resultsof nested two-way ANOVA analysis Material Property Factor F-ValueProbability Stress Treatment 16.060 1.085E−06 Relaxation Fatigue Cycling9.676 2.500E−03 Interaction 1.402 2.515E−01 Hardness Treatment 20.0236.405E−08 Fatigue Cycling 5.898 1.710E−02 Interaction 4.228 1.760E−02

[0043] The data presented above quantifies the elastic and viscoelasticmechanical degradation of intervertebral disc tissue due to repetitive,non-traumatic loading. The results of these experiments establish thatnon-toxic crosslinking reagents reduce the fatigue-related degradationof material properties in a collagenous tissue—namely the intervertebraldisc. More than a three-fold reduction in viscoelastic degradation wasbrought about by soaking the calf disc tissue in 0.33 g/molconcentration of genipin The tested formulation was unable to sustain animprovement in the elastic mechanical properties (hardness) to 3000 testcycles.

[0044] Accurately estimating the length of time it would take an averageperson to experience a comparable amount of wear and tear on theirspinal discs is difficult. Certainly, in addition to the mechanicaldegradation imposed by the described testing, there is theadded—“natural”—degradation of these dead tissues due to the testingenvironment. The non-loaded controls showed this “natural” degradationof material properties to be insignificant. Measures were taken tominimize this natural degradation by keeping the specimens moistthroughout the testing and by accelerating the loading frequency. At thesame time, loading frequency was kept within physiologic limits toprevent tissue overheating. It should be noted that these measuresconstitute standard protocol for in vitro mechanical testing ofcadaveric tissues. Assuming that a person experiences 2 to 20 upright,forward flexion bends per day, these data roughly correspond to severalmonths to several years of physiologic mechanical degradation.

[0045] The described treatment could be repeated at the time periodsrepresented by, for instance, 3000 fatigue cycles at this loadmagnitude. Using the assumption identified above, this number of cyclesmay be estimated to correspond to approximately 1 year for someindividuals. Therefore, with either a single treatment or with repeatedinjections/treatments, an individual may be able to minimize mechanicaldegradation of their intervertebral discs over an extended period oftime. Another option would involve a time-release delivery system suchas a directly applied treated patch, a gel or ointment.

[0046] The invention has been described in terms of certain preferredand alternate embodiments which are representative of only some of thevarious ways in which the basic concepts of the invention may beimplemented. Certain modification or variations on the implementation ofthe inventive concepts which may occur to those of ordinary skill in theart are within the scope of the invention and equivalents, as defined bythe accompanying claims.

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I claim:
 1. A method of improving the resistance of collagenous tissue to mechanical degradation comprising the steps of: contacting at least a portion of a collagenous tissue with an effective amount of a crosslinking reagent.
 2. The method of claim 1, wherein the crosslinking reagent includes genipin.
 3. The method of claim 1, wherein the crosslinking reagent includes proanthrocyanidin.
 4. The method of claim 1, wherein the crosslinking reagent includes a crosslinking agent in a carrier medium.
 5. The method of claim 4, wherein the crosslinking agent is selected from the group consisting of genipin and proanthrocyanidin and the carrier medium is a buffered saline solution.
 6. The method of claim 4, wherein the crosslinking agent is genipin, the carrier medium is a buffered saline solution, and a concentration of the genipin in the buffered saline solution is greater than 0.033%.
 7. The method of claim 1, wherein the collagenous tissue is an intervertebral disc.
 8. The method of claim 1, wherein the collagenous tissue is articular cartilage.
 9. The method of claim 1, wherein the contact between the collagenous tissue and the crosslinking reagent is effected by injections directly into the portion of the collagenous tissue with a needle.
 10. The method of claim 1, wherein the contact between the collagenous tissue and the crosslinking reagent is effected by placement of a time-release delivery system directly into or onto the portion of the collagenous tissue.
 11. The method of claim 10, wherein the time-release delivery system is a gel or ointment.
 12. The method of claim 10, wherein the time-release delivery system is a treated membrane or patch.
 13. The method of claim 10, wherein the time-release delivery system is a treated patch.
 14. The method of claim 1, wherein the contact between the collagenous tissue and the crosslinking reagent is effected by soaking.
 15. The method of claim 1, further comprising the step of: periodically re-contacting the portion of the collagenous tissue with an effective amount of a crosslinking reagent.
 16. A crosslinking reagent comprising a crosslinking agent dissolved in a carrier medium wherein the crosslinking agent is present in an amount effective to improve the fatigue resistance of a portion of a collagenous tissue and wherein the crosslinking agent and the carrier medium are substantially non-cytotoxic.
 17. The crosslinking reagent according to claim 16, wherein the crosslinking agent is selected from the group consisting of genipin and proanthrocyanidin.
 18. A method of improving the resistance of collagenous tissue to mechanical degradation comprising the steps of: preparing a crosslinking reagent by dissolving a crosslinking agent in a carrier medium; and contacting at least a portion of a collagenous tissue with an effective amount of the crosslinking reagent.
 19. The method of claim 18, wherein the contact between the collagenous tissue and the crosslinking reagent is effected by injections directly into the portion of the collagenous tissue with a needle.
 20. The method of claim 18, wherein the contact between the collagenous tissue and the crosslinking reagent is effected by placement of a time-release delivery system directly into or onto the portion of the collagenous tissue.
 21. The method of claim 18, wherein the contact between the collagenous tissue and the crosslinking reagent is effected by soaking.
 22. The method of claim 18, wherein the contact between the collagenous tissue and the crosslinking reagent is effected by spraying.
 23. The method of claim 1, further comprising the step of: periodically re-contacting the portion of the collagenous tissue with an effective amount of a crosslinking reagent. 